I am doing sub cultures of mouse neural stem cells taken from cerebral cortex of E11-13 swiss albino mice embryos.I use the following steps:centrifuge at 800rpm at 4C for 5 min, take cell pellet, add PBS and trypsin edta for 5 min in ice and 5 min in water bath at 37C. add serum , centrifuge at the same speed.Then DNAse digestion for 10min, then centrifuge at the same speed, the add medium, count cells and dilute appropriately and incubate at 37C.

The problem is after DNAse digestion, I am not getting any cell pellet after centrifuging for 5min at 800rpm at 4C. So I am loosing a lot of cells...

Any suggestions?

Thanks

-mdphn-

QUOTE (mdphn @ Jul 17 2007, 09:20 PM)

Hi All,

I am doing sub cultures of mouse neural stem cells taken from cerebral cortex of E11-13 swiss albino mice embryos.I use the following steps:centrifuge at 800rpm at 4C for 5 min, take cell pellet, add PBS and trypsin edta for 5 min in ice and 5 min in water bath at 37C. add serum , centrifuge at the same speed.Then DNAse digestion for 10min, then centrifuge at the same speed, the add medium, count cells and dilute appropriately and incubate at 37C.

The problem is after DNAse digestion, I am not getting any cell pellet after centrifuging for 5min at 800rpm at 4C. So I am loosing a lot of cells...

Any suggestions?

Thanks

how many cells are you expecting from this procedure. You do lose some cells as you proceed thru the process. try counting cells after DNAse treatment and before centifuging it a second time. You will have an idea if you are losing cells or not.

we spin primary cells at 100x g for 5 min at RT.

if you have too less cells, they are very faintly present.

Good Luck !!!

-scolix-

Hi Scolix,

Thanks for the reply, I have been doing the primary neural stem cell culture and subculture for some time now, and i used to get a cell pellet after dnase digestion and centrifugation.But now, after i centrifuge after dnase digestion, there is absolutely no cell pellet. I dont know why, since i am doing the same procedure i have been doing previously. Does the 100rpm spin for 5min at room temp work for neural stem cells too?

-mdphn-

Hi

If what you do is a routine procedure, then now you dont get the cell pellet. Think back at each step to identify any deviation from the normal.

I was only suggesting 100xg as we use it for primary cortical neurons.

Stick with your protocol and try to sort the problem out. I was assuming you were trying it out the first time and were having problems.